Epidermal growth factor receptor (EGFR), which is a transmembrane tyrosine kinase-type receptor, forms a dimer by binding to epidermal growth factor (EGF) or the like, so as to transmit a signal downstream by autophosphorylation activity. This signaling pathway via the EGFR functions, in a normal cell, in control of cell differentiation, cell growth and the like. If hyperactivity occurs in this signaling pathway due to some abnormality, however, the control mechanism for the cell growth and the like fails, which is regarded to cause induction, growth, metastasis, invasion and the like of cancer (Non Patent Literature 1). For example, overexpression of the EGFR is found in about 80% of colorectal cancer (Non Patent Literature 2).
Therefore, as an antibody drug against colorectal cancer based on the aforementioned mechanism, an anti-EGFR antibody drug has recently been developed. For example, cetuximab and panitumumab are anti-EGFR monoclonal antibody drugs for inhibiting the cell growth as a ligand-EGFR binding inhibitor.
It has been revealed, however, that the effect of the anti-EGFR antibody drug such as cetuximab is weak in a patient with KRas gene mutation (Non Patent Literatures 3 and 4). The KRas gene is one of isoforms of the ras gene known as an oncogene. KRAS encoded by this gene activates a signal cascade as a low molecular weight guanosine triphosphate (GTP)-binding protein to transmit downstream a cell growth signal from the epidermal growth factor receptor (EGFR). If a specific position of the KRas gene mutates, the function of the KRAS as GTPase is degraded, and the KRAS becomes a constitutively activated form for continuously transmitting the signal downstream. This excessive signaling is believed to impart a negative effect to the anti-EGFR antibody drug. Actually, in many patients affected with colorectal cancer which tends to become severe, in which the therapeutic effect of the anti-EGFR antibody drug is weak, it has been revealed that mutation is found in codon 12 or codon 13 of the KRas gene (Non Patent Literature 5). Accordingly, it is very significant in selecting a treatment for a patient to precedently predict an effect of the antibody drug by analyzing mutation in KRas gene in a biological sample obtained from the subject before administering the anti-EGFR antibody drug.
However, a pathological sample used in mutation analysis generally contains not only tumor cells but also a large number of normal cells. In particular, in a pathological sample obtained from a subject having early cancer, most of the cells are normal cells and merely a small number of tumor cells having mutant-type KRas gene are mixedly present. Accordingly, there is a serious problem that target mutant-type KRas gene cannot be detected by a usual nucleic acid amplification method because wild-type KRas gene is priorly amplified. Therefore, a technique for simply and sensitively detecting mutant-type KRas gene in the presence of an excessive amount of wild-type KRas gene or a genotype diagnostic agent used as a companion diagnostic agent for precedently determining the efficacy of an anticancer drug is desired.
Therefore, a nucleic acid amplification method for selectively and efficiently amplifying a mutant gene by inhibiting the amplification of a wild-type gene using a nucleic acid analogue and the like has been studied. For example, a method, in which a BNA/LNA (Bridged Nucleic Acid/Locked Nucleic Acid) probe for inhibiting the amplification of a wild-type gene is mixedly used in amplifying a known mutation site by PCR or the like to determine mutation contained in the amplified PCR product by an invader method and the like, has been developed (Non Patent Literature 6). Besides, Patent Literature 1 discloses a method for applying, to PCR clamping, a minor groove binder (MGB)-oligonucleotide conjugate having high affinity with a DNA. Furthermore, Patent Literature 2 and Non Patent Literature 7 describe PCR clamping using PNA (Peptide Nucleic Acid).
Nucleic acid analogs such as BNA/LNA and PNA are, however, expensive, and in addition, there still remains a problem that a combination of the nucleic acid amplification method and the invader method is complicated. Furthermore, all the aforementioned methods still have a problem that the presence of a mutant gene cannot be determined at normal temperature.